A biofilm is a complex and often tenacious aggregate of bacterial cells and secretions adhering to a surface. Biofilms can pose significant threats to human health. In oral biofilms (i.e. dental plaque), for example, the metabolic activity of acid-tolerant bacteria such as Streptococcus mutans generates acid waste products that accumulate near the tooth surface, leading to dental caries. This project focuses on the genetic competence of Streptococcus mutans in oral biofilms. Genetic competence is a cell's ability to take up and incorporate DNA from its environment. It is a key element of S. mutans virulence;it allows S. mutans to acquire new genes for useful traits, so that it can compete more aggressively with other species that inhabit the same biofilm. Competence in the oral streptococci is controlled by complex networks of genes that have been studied extensively but whose operation is still not completely understood: Local environmental factors - physical and chemical conditions like diffusion constants and concentrations of quorum- sensing signals or other chemical products - play a large role in triggering competence. However, the physical and chemical environment varies dramatically from one location to another in a biofilm. A better understanding of genetic competence in S. mutans will require a better understanding of how gene networks respond to complex, heterogeneous environments. This project aims to develop a new experimental approach for unraveling the physical and chemical factors that influence competence and related virulence attributes. We cannot easily manipulate, or even characterize, micro-scale environments inside a natural biofilm. We can however create "artificial biofilms"- microscopic chemical reactors in which we can impose on S. mutans stable and well-defined chemical conditions (concentration gradients, restrictions on physical diffusion, or other chemical and physical conditions) that systematically reproduce aspects of the microscopic environment inside a biofilm. We will use lithographic technology to fabricate microscopic chemostats and other such devices for S.mutans. We will use them to impose controlled spatial and temporal gradients in the signaling molecules that trigger competence, with the goal of identifying local environmental parameters that shape competence response. We will also construct devices that restrict the physical diffusion of signals and cellular products, and thus determine how local biofilm architecture influences cell-to-cell signaling (e.g. quorum-sensing) mechanisms in biofilm-formers like S.mutans. PUBLIC HEALTH RELEVANCE: The bacterium Streptococcus mutans is one of the principle causes of human dental caries. One of the reasons that this organism can successfully colonize surfaces within the human body is that it can take up and use DNA from its environment. The goal of this project is to develop new laboratory devices with which we can study, and perhaps learn to control, this behavior.